Stabilization and activation of protease for use at high temperature

ABSTRACT

Methods for use of select organic compounds for the activation and stabilization of thermally labile enzymes, including proteases, are disclosed. The invention further relates to compositions of certain organic compounds with enzymes to enable the use of such enzymes at high temperature conditions, such as for warewash temperatures and conditions. In particular, organic activator-stabilizers including heat-deactivated proteases and zwitterionic materials are combined with enzymes according to the invention. As a result, the invention provides enzyme cleaning efficacy for various high temperature conditions, including warewash applications.

FIELD OF THE INVENTION

The invention relates to use of enzymes at high temperature conditions,such as ware wash temperatures and conditions in excess of at least 160°F. In particular, the invention relates to use of protease enzymes foreffectively removing soils in warewash applications. The inventionprevents the inactivation and degradation of enzymes, includingproteases, under high temperature conditions as a result of combinationof the enzyme with a stabilizer/activator component. The methods andcompositions according to the invention provide select organic compoundsfor the activation and stabilization of thermally labile enzymes,including proteases. The invention uses select organic compounds,including for example the following organic activator-stabilizers:heat-deactivated proteases and zwitterionic materials, such asamphoteric surfactants and amino acids and proteins.

BACKGROUND OF THE INVENTION

Enzymes are a group of proteins that catalyze a variety of typicalbiochemical reactions and have been employed in cleaning compositionssince early in the 20^(th) century. It was not until the mid 1960's whenenzymes were commercially available with both the pH stability and soilreactivity for detergent applications. Enzymes are known as effectivechemicals for use with detergents and other cleaning agents to breakdown soils. Enzymes break down soils making them more soluble andenabling surfactants to remove them from a surface and provided enhancedcleaning of a substrate.

Enzymes can provide desirable activity for removal of protein-based,carbohydrate-based, or triglyceride-based stains from substrates. As aresult, enzymes have been used for various cleaning applications inorder to digest or degrade soils such as grease, oils (e.g., vegetableoils or animal fat), protein, carbohydrate, or the like. For example,enzymes may be added as a component of a composition for laundry,textiles, cleaning-in-place, drains, floors, carpets, medical or dentalinstruments, meat cutting tools, hard surfaces, personal care, or thelike. The use of enzyme products have evolved from simple powderscontaining alkaline protease to more complex granular compositionscontaining multiple enzymes and still further to liquid compositionscontaining enzymes. In addition, significant progress has been made toobtain enzymes retaining stability under alkaline conditions, such asthe presence of detergent builders and other detergent compositions. Seee.g. U.S. Pat. Nos. 4,771,003, 4,529,525, 4,480,037 and 4,052,262, eachof which are herein incorporated by reference in its entirety. Despitesuch advances there remains a lack of consistent stability under highlyconcentrated conditions for enzyme and detergent formulations and/orlong-term storage of shelf stability.

In addition, there remains a need for cleaning applications usingenzymes under high temperature conditions. Most proteases and otherenzymes are unable to survive at elevated temperatures, typically unableto survive temperatures above about 140° F. to about 150° F. Uponexposure to such elevated temperatures, the enzyme is denatured ordamaged, such that there is a change in the configuration of thestructure (although not limited to a particular theory as one skilled inthe art shall ascertain) and as a result the enzymes are unable to beused in numerous desirable cleaning applications. Enzyme instability dueto decomposition caused by either denaturation or by proteolysis(self-digestion) are well established limitations to the use of enzymesin cleaning compositions. Methods for stabilizing enzymes over extendedperiods of time and for exposure to heat, causing reduced enzymestability, remain as current limitations of enzymes.

Accordingly, it is an objective of the invention to develop methods topermit the use of enzymes to remove soils under high temperatureconditions, such as ware wash applications.

A further object of the invention is to develop methods for stabilizingand activating thermally labile enzymes.

A still further object of the invention is to develop compositions forthe stabilization and activation of thermally labile enzymes for use inhigh temperature conditions, including for example warewashapplications.

BRIEF SUMMARY OF THE INVENTION

An embodiment of the invention includes a method of cleaning a hardsurface comprising: contacting the hard surface with an aqueous usesolution composition comprising from about 0.1 ppm to about 10,000 ppmenzymes, from about 0.1 ppm to about 10,000 ppm organicactivator-stabilizers, and wherein said organic activator-stabilizersprevent the deactivation of said enzymes at temperatures in excess of atleast 150° F.

According to the invention, the methods include the use of a proteaseenzyme and an organic activator-stabilizer that is a heat deactivatedenzyme. The invention further embodies the use of organicactivator-stabilizers selected from the group consisting ofheat-deactivated enzymes, denatured enzymes, zwitterionic materials andcombinations of the same, wherein the zwitterionic materials are membersof the group consisting of amphoteric surfactants, amino acids, proteinsand combinations of the same.

According to additional embodiments of the invention, the proteaseenzyme in an aqueous use solution is between about 1 ppm and about 400ppm, and the heat deactivated enzyme in said aqueous use solution isbetween about 1 ppm and 400 ppm. Further embodiments of the inventionmay include the adfixing of a multi-use solid concentrated compositioncapable of forming the aqueous use solution to the inside of a cleaningapplication (e.g. dishwash or wash apparatus) and contacting a watersource to generate the use solution. Still further, the invention mayinclude the addition of a single use concentrated liquid or solidcomposition capable of forming said aqueous use solution to a watersource to form the aqueous use solution.

An additional embodiment of the invention includes a method of cleaninga hard surface comprising: forming an organic activator-stabilizer;contacting the hard surface with sufficient organic activator-stabilizerto form an aqueous use solution of from about 0.1 ppm to about 10,000ppm organic activator-stabilizer; and contacting the hard surface withsufficient enzyme to generate from about 0.1 ppm to about 10,000 ppmenzymes in the aqueous use solution, wherein said organicactivator-stabilizer prevents the deactivation of said enzymes attemperatures in excess of at least 150° F. The methods may furtherinclude use of a detergent composition, wherein the detergency is notcompromised by the enzymes and/or an organic activator-stabilizer.Preferably, the enzyme is a protease and the organicactivator-stabilizer is a heat deactivated enzyme.

According to an additional embodiment of the invention, the step offorming the organic activator-stabilizer may include exposing an enzymeto sufficient heat to deactivate or denature said enzyme to generatesaid organic activator-stabilizer. The step of forming the organicactivator-stabilizer may be integrated into a cleaning application, suchas adding a first enzyme source to a cleaning application involving awater source at a temperature in excess of about 140° F. to denature theenzyme and form the organic activator-stabilizer.

A still further embodiment of the invention includes an enzymestabilizing and activating aqueous use solution for use at elevatedtemperatures comprising: from about 0.1 ppm to about 10,000 ppm enzymes;and from about 0.1 ppm to about 10,000 ppm organic activator-stabilizersselected from the group consisting of heat-deactivated enzymes,denatured enzymes, zwitterionic materials and combinations of the same,and wherein said organic activator-stabilizers prevent the deactivationof said enzymes at temperatures in excess of at least 150° F. Accordingto particular embodiments of the invention, the enzyme is a protease andthe organic activator-stabilizer is a heat deactivated enzyme.

The particular enzyme or combination of enzymes for use in the methodsand compositions of the invention depend upon the conditions of finalutility, including the physical product form, use pH, use temperature,and soil types to be cleaned in a particular cleaning application. Theenzyme or combination of enzymes are selected to provide optimumactivity and stability for a given set of utility conditions as oneskilled in the art will recognize based on the disclosure of the claimedinvention. In addition, the particular organic activator-stabilizerswill depend upon the selection of the particular enzyme or combinationof enzymes for use as disclosed herein according to the variousembodiments of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The embodiments of this invention are not limited to particular methodsof cleaning using traditionally heat labile enzymes, removing soilsunder high temperature conditions with the use of traditionally heatlabile enzymes and/or compositions for achieving the same beneficialresults, which can vary and are understood by skilled artisans. It isfurther to be understood that all terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting in any manner or scope. For example, as used in thisspecification and the appended claims, the singular forms “a,” “an” and“the” can include plural referents unless the content clearly indicatesotherwise. Further, all units, prefixes, and symbols may be denoted inits SI accepted form. Numeric ranges recited within the specificationare inclusive of the numbers defining the range and include each integerwithin the defined range.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which embodiments of the invention pertain. Many methods andmaterials similar, modified, or equivalent to those described herein canbe used in the practice of the embodiments of the present inventionwithout undue experimentation, the preferred materials and methods aredescribed herein. In describing and claiming the embodiments of thepresent invention, the following terminology will be used in accordancewith the definitions set out below.

The term “about,” as used herein, refers to variation in the numericalquantity that can occur, for example, through typical measuring andliquid handling procedures used for making concentrates or use solutionsin the real world; through inadvertent error in these procedures;through differences in the manufacture, source, or purity of theingredients used to make the compositions or carry out the methods; andthe like. The term “about” also encompasses amounts that differ due todifferent equilibrium conditions for a composition resulting from aparticular initial mixture. Whether or not modified by the term “about”,the claims include equivalents to the quantities refers to variation inthe numerical quantity that can occur.

The term “detersive enzyme,” as used herein, refers to any enzyme havinga cleaning, stain removing or otherwise beneficial effect in cleaningapplications, including for example warewash applications. Enzymes asreferred to herein can be included in the present compositions andmethods of use thereof for a variety of purposes, including for example,removal of protein-based, carbohydrate-based or triglyceride-basedstains or combinations thereof. As disclosed herein, suitable detersiveenzymes (or simply “enzymes” as may also be used herein to describeembodiments of the invention), proteases, amylases, lipases, cellulases,peroxidases and the like and mixtures thereof. The enzymes may be of anysuitable origin, for example bacterial or fungal enzymes.

As used herein, the term “ware” refers to items such as eating andcooking utensils, dishes, and other hard surfaces such as showers,sinks, toilets, bathtubs, countertops, windows, mirrors, transportationvehicles, and floors. As used herein, the term “warewashing” refers towashing, cleaning, or rinsing ware. Ware also refers to items made ofplastic. Types of plastics that can be cleaned with the compositionsaccording to the invention include but are not limited to, those thatinclude polycarbonate polymers (PC), acrilonitrile-butadiene-styrenepolymers (ABS), and polysulfone polymers (PS). Another exemplary plasticthat can be cleaned using the compounds and compositions of theinvention include polyethylene terephthalate (PET).

The term “wash water,” “wash water source,” “wash water solution,” andthe like, as used herein, refer to water sources provided to a cleaningapplication. Wash water or the like, according to the invention, is notlimited according to the source of water. Exemplary water sourcessuitable for use as a wash water source include, but are not limited to,water from a municipal water source, or private water system, e.g., apublic water supply or a well, or any water source. Preferably washwater contains some hardness ions.

The term “weight percent,” “wt-%,” “percent by weight,” “% by weight,”and variations thereof, as used herein, refer to the concentration of asubstance as the weight of that substance divided by the total weight ofthe composition and multiplied by 100. It is understood that, as usedhere, “percent,” “%,” and the like are intended to be synonymous with“weight percent,” “wt-%,” etc.

The compositions and methods described herein according to the inventionprovide the benefit of use of thermally labile enzymes at hightemperature conditions with the use of a stabilizer/activated. Thispresents a significant advantage over prior art cleaning applicationshaving the temperature limitations precluding the use of thermallylabile enzymes, such as proteases. In particular, the invention relatesto use of enzymes at high temperature conditions, such as for ware washtemperatures and conditions in excess of about 160° F. According topreferred embodiments, the invention relates to use of protease enzymesfor effectively removing soils in warewash applications. The inventionprevents the inactivation and degradation of enzymes, includingproteases, under high temperature conditions. The methods andcompositions according to the invention provide select organic compoundsfor the activation and stabilization of thermally labile enzymes,including proteases.

Stabilization and activation of enzymes according to the inventionallows the use of thermally labile enzymes at temperatures exceedingthose suitable for use according to standard temperature limitations,including for example temperatures above at least 150° F./65° C.,preferably above at least about 160° F./71° C., and more preferablyabove at least between about 160-170° F./71-77° C. This overcomes asignificant limitation of the use of the heat labile enzymes havingprior limitations of use above temperatures of approximately 140° F./60°C. In addition to the stabilization of enzymes at high temperaturesaccording to the invention, certain embodiments of the invention furtheractivate the enzymes at the same high temperatures. As a result of thestabilization and/or activation according to the invention and withoutbeing limited to a particular theory of the mechanism of action of thestabilization and/or activation, enzymes are suitable for use at hightemperature ranges. According to a preferred embodiment, thecompositions and methods of use of the invention maintain and/or exceedperformance of the enzymes at temperatures about at least 150° F.,preferably above at least 160° F., and more preferably above at leastbetween 160-170° F.

Compositions

The compositions according to the invention may comprise, consist ofand/or consist essentially of at least one enzyme and at least oneorganic activator-stabilizer. According to further embodiments of theinvention, the compositions may further comprise, consist of and/orconsist essentially of a detergent and/or additional functional agents.

Enzymes

Various enzymes can be used for the compositions according to theinvention to break down adherent soils, such as starch or proteinaceousmaterials, typically found in soiled surfaces and typically treated withdetergent compositions. Various enzymes for use according to theinvention to decrease and/or eliminate the soils on a treated substratemay be utilized in combination with the organic activator-stabilizercompounds.

Exemplary types of enzymes which can be incorporated into thecompositions according to the invention include protease, amylase,lipase, esterase, cellulase, cutinase, gluconase, peroxidase and/ormixtures thereof. The compositions according to the invention may employmore than one enzyme, from any suitable origin, such as vegetable,animal, bacterial, fungal or yeast origin. According to an embodiment ofthe invention, the composition includes at least one enzyme. Accordingto a further embodiment of the invention, the composition includes atleast two enzymes. According to a still further embodiment of theinvention, mixtures of the same class of enzymes are incorporated intothe compositions, such as a mixture of various protease enzymes.

Examples of commercially-available protease enzymes are available underthe following trade names: Savinase, Esperase, Purafect, Purafect L,Purafect Ox, Everlase, Liquanase, Prime L, Prosperase and Blap. Examplesof commercially-available amylase enzymes are available under thefollowing trade names: Purastar, Purastar ST, HP AmL, Maxamyl, Duramyl,Termamyl and Stainzyme. Lipases are commercially available, for example,under the trade name Lipex and Lipolase. Cellulase enzymes arecommercially-available, for example, under the trade name Celluzyme.

According to the invention, the enzyme(s) selected for a particularcomposition may be varied based on the particular cleaning applicationand the types of soils in need of cleaning According to the invention,the temperature of a particular cleaning application is no longer alimiting factor for the selection of enzymes as a result of thecombination of the enzyme with the select organic activator-stabilizercompounds. According to a preferred embodiment of the invention, forware wash applications which require temperatures in excess of about150° F. proteases are desirable.

According to the invention, the active level of enzyme in the aqueoususe solution may be modified according to the precise requirements ofthe cleaning application. For example, the amount of enzyme formulatedinto the enzyme composition may vary. Alternatively, as one skilled inthe art will appreciate, the active level of the aqueous use solutionmay be adjusted to a desired level through control of the wash time,water temperature at which the water source contacts the enzymecomposition or the enzyme and detergent composition in order to form theaqueous use solution and the detergent selection and concentration.

The amounts of enzymes according to the invention constitute acleaning-effective amount, and refer to any amount capable of producinga cleaning, stain removal, soil removal, whitening, deodorizing orfreshness improving effect on substrates, including for examplewarewash. According to a preferred embodiment, an aqueous use solutionaccording to the invention may comprise, consist of and/or consistessentially of approximately 0.1 ppm and 10,000 ppm enzyme, preferablybetween about 0.5 ppm and about 800 ppm, and more preferably betweenapproximately 1 ppm and 400 ppm enzyme.

According to further embodiments of the invention, the amount of enzymeneeded to clean and remove soils from a particular cleaning applicationvaries according to the type of cleaning application and the soilsencountered in such applications. According to various embodiments ofthe invention, levels of enzymes in an aqueous use solution areeffective at or below approximately 0.1 ppm, 0.5 ppm or 1 ppm. Accordingto alternative embodiments, use levels of enzymes may be as great as10,000 ppm, with most applications utilizing enzymes in aqueous usesolutions between approximately 1-400 ppm.

In addition to variations in the concentration of enzymes incompositions according to the invention, as one skilled in the art shallascertain, enzymes are further selected based upon the ability to cleanspecific types of soils. A variety of detersive enzymes can be usedaccording to the invention. For example, according to an embodiment ofthe invention, ware wash applications may use an amylase enzyme as it isin reducing starchy, carbohydrate-based soils. Although not limiting thepresent invention, it is believed that amylase can be advantageous forcleaning soils containing starch. Amylase enzymes can be obtained fromany suitable source, such as bacterial strains, barley malt, certainanimal glandular tissues and any others known to the art. Amylaseenzymes may include those which are referred to as alpha-amylases,beta-amylases, iso-amylases, pullulanases, maltogenic amylases,amyloglucosidases, and glucoamylases, as well as other amylases enzymesnot particularly identified herein. These also include endo- andexo-active amylases.

According to an alternative embodiment, methods of cleaning wareapplications may further use a protease to reduce starch, protein andoil soils. According to an additional alternative embodiment of theinvention, a combination of amylase and protease enzymes may be used forcleaning applications employing a detergent in order to most effectivelyprevent starch, proteins and oils from hindering detergent performance.Although not limiting the present invention, it is believed thatprotease can be advantageous for cleaning soils containing protein, suchas blood, cutaneous scales, mucus, grass, food (e.g., egg, milk,spinach, meat residue, tomato sauce), or the like. Protease enzymes arecapable of cleaving macromolecular protein links of amino acid residuesand convert substrates into small fragments that are readily dissolvedor dispersed into a wash water source. Proteases are often referred toas detersive enzymes due to the ability to break soils through thechemical reaction known as hydrolysis. Protease enzymes can be obtained,for example, from Bacillus subtilis, Bacillus licheniformis andStreptomyces griseus. Protease enzymes are also commercially availableas serine endoproteases.

According to an additional embodiment of the invention, a cellulose orlipase enzyme may be incorporated into a composition. Although notlimiting the present invention, it is believed that cellulase can beadvantageous for cleaning soils containing cellulose or containingcellulose fibers that serve as attachment points for other soil.Although not limiting to the present invention, it is believed thatlipase enzymes can be advantageous for cleaning soils containing fat,oil, or wax, such as animal or vegetable fat, oil, or wax (e.g., saladdressing, butter, lard, chocolate, lipstick). Both cellulase and lipaseenzymes can be derived from a plant, an animal, or a microorganism, suchas a fungus or a bacterium. A cellulase or lipase enzyme can be purifiedor a component of an extract, and either wild type or variant (eitherchemical or recombinant).

Additional enzymes suitable for certain embodiments of the inventioninclude cutinase, peroxidase, gluconase, and the like. Suitable enzymesare described for example in WO 8809367 (cutinase), WO 89099813 and WO8909813 (peroxidases), and WO 9307263 and WO 9307260 (gluconase). Knownperoxidase enzymes include horseradish peroxidase, ligninase, andhaloperoxidases such as chloro- or bromo-peroxidase. Peroxidase enzymescan be used in combination with oxygen sources, e.g., percarbonate,perborate, hydrogen peroxide, and the like. Each of these enzymes may bederived from a plant, an animal, or a microorganism and can be purifiedor a component of an extract, and either wild type or variant (eitherchemical or recombinant).

Additional description of enzyme compositions suitable for use accordingto the invention is disclosed for example in U.S. Pat. Nos. 7,670,549,7,723,281, 7,670,549, 7,553,806, 7,491,362, 6,638,902, 6,624,132,6,569,827, 6,197,739 and U.S. patent application Ser. No. 12/642,091filed Dec. 18,2009 titled “Multiple Enzyme Cleaner for SurgicalInstruments and Endoscopes,” Ser. No. 11/279,654, filed Apr. 13, 2006titled “Stable Solid Compositions of Spores, Bacteria, Fungi and/orEnzyme,” Ser. No. 10/923,420, filed Aug. 20, 2004 titled “Enzyme-BasedCleaning Composition and Method of Use,” Ser. No. 10/654,333, filed Sep.2, 2003 titled “Stable Solid Enzyme Compositions and Methods EmployingThem,” the contents of which are incorporated by reference in itsentirety. In addition, the reference “Industrial Enzymes”, Scott, D., inKirk-Othmer Encyclopedia of Chemical Technology, 3rd Edition, (editorsGrayson, M. and EcKroth, D.) Vol. 9, pp. 173-224, John Wiley & Sons, NewYork, 1980 is incorporated herein in its entirety.

Organic Activator-Stabilizers

According to the invention, select organic compounds can both activateand stabilize enzymes for use in high temperature conditions, such asware wash applications. According to an embodiment of the invention, thefollowing organic activator-stabilizers can be employed:heat-deactivated or denatured enzymes and zwitterionic materials, suchas amphoteric surfactants and amino acids and proteins.

Damaged or Denatured Enzyme

According to an embodiment of the invention, an organicactivator-stabilizer for use in compositions of the invention mayinclude a damaged or denatured enzyme. Without being limited to aparticular theory of the invention, it is understood that variations inthe ambient temperature, pH, salinity and/or other conditions negativelyimpact the stability of a protein. Accordingly, as a skilled artisanwill appreciate, changes to the temperature, pH, salinity, etc. of anenzyme can result in the denaturation of the enzyme. Denaturation ordamage to an enzyme is understood to negatively impact the threedimensional confirmation of the enzyme. As one skilled in the art willascertain and without being limited to a particular mechanism of action,damaging an enzyme is one mechanism for obtain an organicactivator-stabilizer for use in the compositions according to theinvention.

It is highly unexpected that the inactivation or denaturation of anenzyme would have the effect of stabilizing and/or activating anotherenzyme (a non-inactivated or non-denatured enzyme) when used incombination. One theory of the invention yielding these unexpectedresults, is that the damaged enzyme frees calcium ions from the threedimensional structure and assists in activating the non-damaged enzymeyielding an enzyme source that is activated (or at least maintainsactivity) at high temperature conditions.

According to one embodiment of the invention, heat-deactivated enzymes,namely proteases, can be used as the organic activator-stabilizeraccording to the invention. According to a non-limiting embodiment ofthe invention, a protease may be treated with sufficient heat and for asufficient period of time in order to deactivate the enzyme. Thereafter,the deactivated or denatured protease may be combined with anotherenzyme for purposes of acting as an activator-stabilizer. For example,an enzyme may be heat deactivated by exposing the enzyme to heat aboveambient temperature and below about 170° F., preferably above about 120°F., and more preferably above about 140° F. The enzyme may be exposed tothe heat for a few minutes, preferably about 5 minutes to cause thedeactivation or denaturation.

As one skilled in the art will ascertain there are alternativemechanisms to deactivate a protease and other enzymes. The compositionsof the invention as set forth herein are not limited in scope by the useof a particular mechanism for the deactivation of an enzyme or theparticular enzyme to be deactivation for use as the organicactivator-stabilizer according to the invention. However, according to apreferred embodiment of the invention, at least one protease enzyme isdeactivated using heat for use as the organic activator-stabilizeraccording to the invention.

Zwitterionic Materials

According to an embodiment of the invention, an organicactivator-stabilizer for use in compositions of the invention mayinclude at least one zwitterionic material. Examples of suitablezwitterionic materials for use according to the invention as an organicactivator-stabilizer include amphoteric surfactants, amino acids and/orproteins.

Examples of suitable amphoteric surfactants according to the inventioninclude, for example, betaines, amine oxide, sulfbetaines and sultaines.

Examples of suitable amphoteric amino acids according to the inventioninclude, for example, amino acids with electrically charged side chains.Although not intending to limit the invention in scope, electricallycharged side chains can include, for example positive charges, such asfor example, arginine, histidine and lysine; negative charges, such asfor example, aspartic acid and glutamic acid; polar uncharged sidechains, such as serine, threonine, asparagine and glutamine. Inaddition, amino acids with hydrophobic side chains are suitableamphoteric amino acids according to the invention for use as an organicactivator-stabilizer.

Examples of suitable amphoteric proteins according to the inventioninclude, for example, any protein having a free carboxylic and/or freeamino group at the end of the protein that is able to react with acidsand/or bases (dependent upon acidic or alkaline mediums). For example, awhey protein may be a suitable organic activator-stabilizer according toan embodiment of the invention.

According to the invention, the active level of a particular organicactivator-stabilizer compound or compounds in the aqueous use solutionmay be modified according to the precise requirements of the cleaningapplication. For example, the amount of organic compounds formulatedinto the enzyme composition may vary. Alternatively, as one skilled inthe art will appreciate, the active level of the aqueous use solutionmay be adjusted to a desired level through control of the wash time,water temperature at which the water source contacts the enzymecomposition or the enzyme and detergent composition in order to form theaqueous use solution and the detergent selection and concentration.

According to a preferred embodiment, an aqueous use solution accordingto the invention may comprise, consist of and/or consist essentially ofapproximately 0.1 ppm and 1000 ppm organic activator-stabilizercompounds, preferably between about 0.5 ppm and about 800 ppm, and morepreferably between approximately 1 ppm and 400 ppm organicactivator-stabilizer compounds.

According to further embodiments of the invention, the amount of organicactivator-stabilizer compounds needed to activate and stabilize enzymesto clean and remove soils from a particular cleaning application variesaccording to the type of cleaning application and the soils encounteredin such applications. According to various embodiments of the invention,levels of organic activator-stabilizer compounds in an aqueous usesolution are effective at or below approximately 0.1 ppm, 0.5 ppm or 1ppm. According to alternative embodiments, use levels of organicactivator-stabilizer compounds may be as great as 10,000 ppm, with mostapplications utilizing organic activator-stabilizer compounds in aqueoususe solutions between approximately 1-400 ppm.

Detergents

The compositions according to the invention may be an independent entityand/or may be formulated in combination with a detergent composition.According to an embodiment of the invention, a composition may beformulated into a detergent composition in either liquid or solidformulations. In addition, compositions may be formulated into variousdelayed or controlled release formulations.

Detergent Compositions

According to the invention, the detergent composition may be liquids orsolids, including for example molded compositions, as are appreciated bythose skilled in the art. Pastes and gels can be considered types ofliquid. Powders, agglomerates, pellets, tablets, and blocks can beconsidered types of solid. For example, detergent compositions may beprovided in the form of blocks, pellets, powders (i.e., mixture ofgranular dry material), agglomerates and/or liquids under roomtemperature and atmosphere pressure conditions. Powder detergents areoften prepared by mixing dry materials or by mixing a slurry and dryingthe slurry. Pellets and blocks are typically provided with a size thatis determined by the shape or configuration of the mold or extruderthrough which the detergent composition is compressed. Pellets aregenerally characterized as having an average diameter of about 0.5 cm toabout 2 cm. Blocks are generally characterized as having an averagediameter of greater than about 2 cm, preferably between about 2 cm andabout 2 ft., and can have an average diameter of between about 2 cm andabout 1 ft. According to a preferred embodiment, a solid block is atleast 50 grams.

According to certain embodiments of the invention, the detergentcomposition is substantially free of phosphorous. Substantiallyphosphorous-free refers to a composition to which phosphorous-containingcompounds are not added. In an exemplary embodiment, the cleaningcomposition includes less than approximately 10% phosphates,phosphonates, and phosphites, or mixtures thereof by weight. Preferably,the detergent composition includes less than approximately 5%phosphates, phosphonates, and phosphites by weight. More preferably, thedetergent composition includes less than approximately 1% phosphates,phosphonates, and phosphites by weight. Most preferably, the detergentcomposition includes less than approximately 0.1% phosphates,phosphonates, and phosphites by weight.

Additional description of detergent compositions, and methods offormation of the same, suitable for use according to the invention aredisclosed, for example, in U.S. Pat. Nos. 7,674,763, 7,153,820,7,094,746 and 6,924,257 and U.S. patent application Ser. No. 12/695,370,filed Jan. 28, 2010 titled “Method for Washing an Article using a MoldedDetergent Composition,” the contents of which are incorporated byreference in its entirety.

Use of detergent compositions with the aqueous use solution according tothe invention can be used in conventional detergent dispensingequipment. For example, commercially available detergent dispensingequipment which can be used according to the invention are availableunder the name Solid System™ from Ecolab, Inc. Use of such dispensingequipment results in the erosion of a detergent composition by a watersource to form the aqueous use solution according to the invention.

Additional Functional Ingredients

One skilled in the art will ascertain that additional functionalingredients may be used with the compositions according to theinvention, such that the ingredients are compatible with the cleaningcompositions. The term “compatible,” as used herein, means theadditional functional ingredients do not reduce and/or otherwisenegatively impact the efficacy of the composition, including theenzymatic activity of the protease or other enzymes, to such an extentthat the enzyme is not effective as desired during its intended useaccording to the methods of the present invention.

The compositions of the invention may also comprise, consist of and/orconsist essentially of stabilizers, buffers, cofactors, inert vehicles,solvents, dyes, fragrances, anti-redeposition agents, corrosioninhibitors, defoamers, antimicrobial agents, preservatives, chelators,bleaching agents and combinations of the same.

Exemplary aesthetic additives which can be used as additional componentsinclude dyes and fragrances, such as dye #2, and a preferred fragranceincludes lemon fragrance. Exemplary anti-redeposition agents which canbe incorporated according to the invention include sodium carboxymethylcellulose, sodium polyacrylate, and hydroxypropyl cellulose.Exemplary corrosion inhibitors which can be incorporated according tothe invention include triethanolamine, and doderylamine. Numerousadditional corrosion inhibitors can be incorporated and are described,for example, in U.S. patent application Ser. No. 12/617,419, filed Nov.12, 2009 titled “Warewashing Composition for Use in AutomaticDishwashing Machines, and Methods for Manufacturing and Using,” thecontents of which are incorporated by reference in its entirety.Additional anti-etch agents can be further utilized to reduce theetching or corrosion found on certain surfaces treated with detergentcompositions. Examples of suitable anti-etch agents include adding metalions to the composition such as zinc, zinc chloride, zinc gluconate,aluminum, and beryllium. However, according to certain embodiments ofthe invention, anti-etch agents are not required for use of the methodsof the present invention.

Exemplary buffering agents which can be incorporated according to theinvention include sodium acetate, potassium dihydrogen phosphate, andsodium borate. Exemplary defoamers which can be incorporated accordingto the invention include polymeric silicone derivatives, and alkynolderivatives. Exemplary antimicrobial agents which can be incorporatedmay include paraben materials such as propyl paraben. Additionalantimicrobial agents which can be incorporated according to theinvention include tert-amylphenol, quaternary ammonium compounds, andactive halogen containing compounds. Exemplary chelators which can beincorporated according to the invention include nitrilotriacetic acid(NTA) and ethylenediaminetetraacetic acid (EDTA) to help control scale,remove soils, and/or sequester metal ions such as calcium, magnesium andiron.

Bleaching agents may also be incorporated according to the invention inorder to lighten or whiten a substrate, and can include bleachingcompounds capable of liberating an active halogen species, such as Cl₂,Br₂ —OCl — and/or —OBr—, or the like, under conditions typicallyencountered during the cleansing process. Examples of suitable bleachingagents include, but are not limited to: chlorine-containing compoundssuch as chlorine, a hypochlorite or chloramines. Examples of suitablehalogen-releasing compounds include, but are not limited to: alkalimetal dichloroisocyanurates, alkali metal hypochlorites, monochloramine,and dichloroamine. Encapsulated chlorine sources may also be used toenhance the stability of the chlorine source in the composition (see,for example, U.S. Pat. Nos. 4,618,914 and 4,830,773, the disclosures ofwhich are incorporated by reference herein). The bleaching agent mayalso include an agent containing or acting as a source of active oxygen.The active oxygen compound acts to provide a source of active oxygen andmay release active oxygen in aqueous solutions. An active oxygencompound can be inorganic, organic or a mixture thereof. Examples ofsuitable active oxygen compounds include, but are not limited to:peroxygen compounds, peroxygen compound adducts, hydrogen peroxide,perborates, sodium carbonate peroxyhydrate, phosphate peroxyhydrates,potassium permonosulfate, and sodium perborate mono and tetrahydrate,with and without activators such as tetraacetylethylene diamine. It isto be appreciated by a skilled artisan that certain embodiments of theinvention preferably use compositions that are chlorine-free to promotethe use of enzymes according to the invention.

In a further embodiment of the invention, a bleach or any bleachingcomponent is not required as part a component of the compositions and/ormethods of the invention.

One skilled in the art shall ascertain additional components that may beused in combination with the methods of the present invention.

Additional Enzyme Stabilizers

The compositions for use in the methods of the present invention mayfurther include enzyme stabilizers. One skilled in the art willascertain suitable enzyme stabilizers and/or stabilizing systems forenzyme compositions suitable for use according to the invention, and mayinclude those described, for example, in U.S. Pat. Nos. 7,569,532 and6,638,902, which are incorporated herein by reference in their entirety.According to an embodiment of the invention, an additional enzymestabilizing system may include a mixture of carbonate and/or bicarbonateand can also include other ingredients to stabilize certain enzymes orto enhance or maintain the effect of the mixture of carbonate andbicarbonate. An enzyme stabilizer may further include boron compounds orcalcium salts. For example, enzyme stabilizers may be boron compoundsselected from the group consisting of boronic acid, boric acid, borate,polyborate and combinations thereof.

According to an embodiment of the invention, the additional enzymestabilizers do not include chlorine bleach scavengers for the preventionof chlorine bleach species attacking and inactivating the enzymes (e.g.alkaline conditions).

According to alternative embodiments of the invention, the enzymecompositions for use in the methods of the present invention arepreferably free of additional enzyme stabilizers. According to apreferred embodiment, the enzyme compositions are free of anyenzyme-stabilizing calcium and/or magnesium sources.

Composition Formulations

The enzyme (or enzymes) and organic activator-stabilizer compounds canbe formulated into a variety of compositions according to the invention.All formulations disclosed herein shall be understood to optionallyinclude the additional composition components disclosed according to theinvention, including for example the detergents, any combination ofadditional functional ingredients and/or additional enzyme stabilizers.The description of composition formulations suitable for use in themethods of the invention shall not be limited according to theparticular formulations described herein.

According to an embodiment of the invention, the compositions accordingto the invention may be incorporated into a solid cleaning composition.Exemplary solid cleaning composition include a solid form such as apowder, a flake, a granule, a pellet, a tablet, a lozenge, a puck, abriquette, a brick, a solid block, a unit dose, or another solid formknown to those of skill in the art. The term “solid” refers to the stateof the composition under the expected conditions of storage and use ofthe solid composition. In general, it is expected that the solidcomposition will remain in solid form when exposed to temperatures of upto about 100° F. and greater than about 120° F.

According to an embodiment of the invention, the compositions accordingto the invention may be incorporated into a solid or a liquidconcentrate composition. According to an embodiment of the invention asolid or liquid concentrate can be provided in a suitable formulation tobe adfixed inside a washer for multiple use applications. Formulationsfor multiple-use solids, such as, a block or a plurality of pellets, andcan be repeatedly used to generate aqueous compositions for multiplewashing cycles. In certain embodiments, the solid multi-use compositionis provided as a solid having a mass of about 5 g to 10 kg. In certainembodiments, a multiple-use form of the solid composition has a mass ofabout 1 to 10 kg. In further embodiments, a multiple-use form of thesolid composition has a mass of about 5 kg to about 8 kg. In otherembodiments, a multiple-use form of the solid composition has a mass ofabout 5 g to about 1 kg, or about 5 g and to 500 g.

For example, a solid or liquid concentrate composition may be comprisedof at least one enzyme source, at least one organic activator-stabilizercompound source (e.g. heat-deactivated enzymes) and optionally adetergent source. According to an alternative embodiment of theinvention, a solid or liquid concentration composition can be providedin a formulation suitable for adfixing inside a washer or otherapparatus and may be comprised of the organic activator-stabilizercompound source (e.g. heat-deactivated enzymes) and other optionalfunctional ingredients. According to such an embodiment, the enzymesource (non-deactivated) is added during the cleaning application asfurther described according to the methods of use of the presentinvention.

In certain embodiments, a solid composition is provided in the form of aunit dose. A unit dose refers to a solid cleaning composition unit sizedso that the entire unit is used during a single cleaning cycle, such asa washing cycle. When the solid composition is provided as a unit dose,it can have a mass of about 1 g to about 50 g. In other embodiments, thecomposition can be a solid, a pellet, or a tablet having a size of about50 g to 250 g, of about 100 g or greater, or about 40 g to about 11,000g.

For purposes of clarity, according to various embodiments of theinvention wherein the organic activator-stabilizer compound is apre-treated enzyme source (e.g. heat-deactivated enzymes), thecompositions will have at least two distinct enzyme compositions in theformulation: a pre-treated enzyme source suitable for acting as theorganic activator-stabilizer compound and a second enzyme source(non-deactivated), wherein the second enzyme source is activated and/orstabilized according to the methods of the invention as a result of thecombination with the pre-treated enzyme source.

According to a further embodiment of the invention, the compositionsaccording to the invention may be obtained commercially in a solid(i.e., puck, powder, etc.) or liquid formulation. According to analternative embodiment of the invention the compositions can beincorporated into a detergent composition.

According to a still further alternative embodiment of the invention thecompositions can be formulated and used as separate product systems. Forexample, an enzyme source can be separate from an organicactivator-stabilizer source and combined at a point of use for aparticular cleaning application as described further according to themethods of use of the present invention.

The methods of manufacture of the various compositions formulationsdisclosed herein are well known to a skilled artisan and such methods ofmanufacture are not critical to the present invention.

Packaging Systems

In some embodiments, the solid formulations can be packaged. Thepackaging receptacle or container may be rigid or flexible, and composedof any material suitable for containing the compositions producedaccording to the invention, as for example glass, metal, plastic film orsheet, cardboard, cardboard composites, paper, and the like. A mixturemay be formed directly in the container or other packaging systemwithout structurally damaging the material. As a result, a wider varietyof materials may be used to manufacture the container than those usedfor compositions that processed and dispensed under molten conditions.

Suitable packaging used to contain the compositions is manufactured froma flexible, easy opening film material.

Methods of Use

According to an embodiment of the invention, enzyme stabilizing andactivating compositions are used to provide improved cleaning throughthe activated use of enzymes under high temperatures providing improveddetergency. Enzymes are used according to the methods of the inventionto effectively remove soils from a variety of substrates. In addition,the methods of using activated and stabilized enzymes for cleaningapplications at high temperatures further promotes cleaning of varioussurfaces, including ware and the wash equipment surfaces itself, such asthe interior of a washing machine by improving the detergency of thecleaning application. The various methods of use according to theinvention present a significant advantage over prior art cleaningmethods having significant temperature limitations that effectivelypreclude the use of thermally labile enzymes, such as proteases.

Dispensing of the Compositions

The cleaning composition according to the present invention can bedispensed in any suitable method generally known. The cleaning orrinsing composition can be dispensed from a spray-type dispenser such asthat disclosed in U.S. Pat. Nos. 4,826,661, 4,690,305, 4,687,121,4,426,362 and in U.S. Pat. Nos. Re 32,763 and 32,818, the disclosures ofwhich are herein incorporated by reference in their entirety. Briefly, aspray-type dispenser functions by impinging a water spray upon anexposed surface of the solid composition to dissolve a portion of thecomposition, and then immediately directing the concentrate solutionincluding the composition out of the dispenser to a storage reservoir ordirectly to a point of use. When used, the product is removed from thepackage (e.g.) film and is inserted into the dispenser. The spray ofwater can be made by a nozzle in a shape that conforms to the solidshape. The dispenser enclosure can also closely fit the detergent shapein a dispensing system that prevents the introduction and dispensing ofan incorrect detergent. The aqueous concentrate is generally directed toa use locus.

In an embodiment, the present composition can be dispensed by immersingeither intermittently or continuously in water. The composition can thendissolve, for example, at a controlled or predetermined rate. The ratecan be effective to maintain a concentration of dissolved cleaning agentthat is effective for cleaning

In an embodiment, the present composition can be dispensed by scrapingsolid from the solid composition and contacting the scrapings withwater. The scrapings can be added to water to provide a concentration ofdissolved cleaning agent that is effective for cleaning

Methods Employing the Compositions

One skilled in the art will appreciate that the methods according to theinvention can be used for a variety of cleaning applications, forenhanced performance and/or reducing cleaning costs. The variouscleaning applications may include for example, ware washing, instrumentcleaning, manual pot and pan cleaning, presoak products, drain cleaning,food and beverage plant cleaning, housekeeping applications and thelike. Additional cleaning applications according to the methods of theinvention may include laundry washing and other applications. Forexample, laundry applications according to the invention may include theuse of the compositions with detergents, presoaks, rinse and cleaners,sours, softeners and the like.

Still further, cleaning applications in health care may further benefitfrom the methods according to the invention, including for example,various instruments and surfaces used in health care facilities. Inaddition, cleaning applications in commercial restaurants, housekeepingand like may further employ methods of the present invention as oneskilled in the art shall ascertain.

Beneficially, the methods of the invention allow the use of enzymes athigh temperature conditions, such as for warewash temperatures andconditions. According to particular embodiment, the methods of theinvention permit the use of various heat labile enzymes at temperaturesabove at least 150° F., preferably above at least 160° F., and morepreferably above at least 160-170° F. This overcomes a significantlimitation of the use of the heat labile enzymes having priorlimitations of use above temperatures of approximately 60° C. (140° F.).

The enzyme (or enzymes) and organic activator-stabilizer compounds canbe provided to cleaning application in a variety of formulations,including for example, solid or a liquid concentrates. The methods ofthe invention described herein are not limited to a particularformulation or composition according to the invention. Exemplary methodsare described and one skilled in the art will ascertain that the variousformulations disclosed can be used according to the various methods ofuse.

In some embodiments, the methods of the invention include a pretreatmentstep to obtain the organic activator-stabilizer compound. In the eventthe organic activator-stabilizer is a deactivated enzyme, thepretreatment step may include subject a source of enzymes to variationsin the ambient temperature, pH, salinity and/or other conditions thatare expected to negatively impact the stability of a protein. Thepretreatment steps to cause denaturation or damage to the enzymespreferably include the use of heat to deactivate the enzymes, namelyproteases, for use as the organic activator-stabilizer according to theinvention. According to a non-limiting embodiment of the invention, theenzymes are pretreated with sufficient heat and for a sufficient periodof time in order to deactivate the enzyme. For example, an enzyme may beheat deactivated by exposing the enzyme to heat above ambienttemperature and below about 170° F., preferably above about 120° F., andmore preferably above about 140° F. The enzyme may be exposed to theheat for a few minutes, preferably about 5 minutes to cause thedeactivation or denaturation.

According to alternative embodiments of the invention, a pretreatmentstep is not employed with alternative organic activator-stabilizercompounds are utilized in a particular composition (i.e. non-deactivatedenzymes). One skilled in the art will ascertain that the method of firstdeactivating an enzyme source for subsequent use and/or combination intoa formulation with an additional enzyme source and optionally adetergent source provides for methods of use that significantly divertfrom the methods employed by those skilled in the art. It is unexpectedthat by first deactivating a source of enzymes (such as through the useof heat) will result in the activation and/or stabilization of a secondenzyme source for enhanced use and performance in cleaning applicationsunder high temperatures.

According to an embodiment of the invention the solid or liquidconcentrate contacts a wash water source. The contacting step mayinclude the adfixing of the composition to the inside of a washer formultiple use applications. Alternatively, the compositions can beincorporated into detergent compositions and introduced to a wash watersource for a particular cleaning application as one skilled in the artwould ascertain. Still further, alternative methods of contacting thecompositions of the invention with a wash water source may include theintroduction of the composition elements independently or in sequence.For example, the organic activator-stabilizer compounds (e.g.deactivated enzymes) may be first introduced to a cleaning process,wherein the second enzyme source is subsequently introduced. One skilledin the art will ascertain that the timing of introduction of thecompositions is an important aspect of the invention. For example, it isimportant for the organic activator-stabilizer to be first introduced toensure that the addition of the enzymes are activated and stabilizedunder the high temperature conditions.

According to another embodiment, the organic activator-stabilizer isadded at least 1 minute prior to the second enzyme source. In anotherembodiment, the organic activator-stabilizer is added to and maintainedin a sump solution of washing machine prior to the addition of thesecond enzyme source. One skilled in the art will ascertain additionalembodiments of the application of the organic activator-stabilizeraccording to the invention.

The compositions may be introduced, for example, manually or by adispenser, pump, pump and control system or other means. According tothe invention, an aqueous use solution is generated by adding thecompositions of the invention to a water source. According to variousembodiments of the invention, an aqueous use solution may also begenerated by adding the compositions of the invention to a furtherdetergent composition. Alternatively according to the invention, adetergent composition may be formulated in combination or separatelyfrom the organic activator-stabilizer compound and enzyme composition.

The methods of using activated and stabilized enzyme compositionsaccording to the invention include cleaning a soiled substrate, such aswarewash applications. According to embodiments of the invention,enzymes reduce or eliminate soil content in a wash application or on asubstrate surface. Preferred embodiments of the invention providecomplete elimination of soil levels in a wash application or on asubstrate surface. According to a further embodiment of the invention,methods of cleaning using activated and stabilized enzymes furtherresult in the prevention of and removal of soil buildup on the interiorsurfaces of cleaning equipment and treated surfaces contained therein.Such surfaces may be either removable or permanent surfaces of cleaningequipment.

All publications and patent applications in this specification areindicative of the level of ordinary skill in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated by reference.

EXAMPLES

Embodiments of the present invention are further defined in thefollowing non-limiting Examples. It should be understood that theseExamples, while indicating certain embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theembodiments of the invention to adapt it to various usages andconditions. Thus, various modifications of the embodiments of theinvention, in addition to those shown and described herein, will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

Example 1

Photographic film contains a layer of a binding agent that is a protein(gelatin) which is readily attacked and removed by protease underambient conditions. The film has multiple colored layers of the gelatinbinding agent which can serve as an indication of the degree of attackif exposed to suitable enzymes. Photographic film was used inexperiments to show the ability of enzymes, proteases, to attach orremove the protein layer under various conditions. In particular, theremoval of the various layers of protein results in different colorsremaining on the film, as set forth herein: tan indicates no attack (orchange) of the protein layer by the enzyme; red indicates the proteinhas removed the first layer; and blue indicates that the enzyme removedthe first and second layers of the protein on the photographic film. Asset forth in Table 1, bolded results indicate that the enzyme removedboth the first and second layers of the protein on the film, yielding ablue result on the photographic film.

In the experiments outlined in Table 1, a beaker was charged with 5grain tap water. An activator, “Additive 1”, was added to the beakerwith the water temperature adjusted to the temperature as shown in the“Temp (F) Additive 1” column. The solution was then adjusted for thefilm test to the temperature as shown in “Temp (F) Enzyme” and aprotease enzyme and section of photographic film added. The solution wasstirred for 5 minutes and the film recovered and wiped with a papertowel for visual examination.

Protease enzymes used for this Example included Savinase and Esperase(tradenames available from Novozyme). The lipase enzyme used for thisExample is Lipex (tradename also available from Novozyme).

The additive, enzyme and then the combination of protease enzyme andadditives were heated to the temperatures indicated in the applicablecolumns to analyze the various conditions under which the proteaseenzyme was able to readily attack and removed the protein (gelatin)layers of the photographic film.

The “Additive 1” and “Temp (F) Additive 1” columns indicates (ifapplicable) the concentration of additive added to a beaker of water andtemperature of the water at the time of addition of a particularadditive to the test beaker. The solution was then heated forapproximately 5 minutes to reach the temperature shown in the “Temp (F)Enzyme” column. The test enzyme and photographic film were then added tothe solution in an amount sufficient to obtain the particularconcentration of enzyme in the test beaker that shown in the “Enzyme”column. The concentration of test enzyme added at this point is shownunder the heading “Enzyme”. The temperature was held for a sufficientperiod of time at the temperature shown in the “Temp (F) Film Treatment”column. The strip was stirred in the solution and then removed after 5minutes. Any strip was wiped with a paper cloth to remove any loose(e.g. digested) gelatin.

The items in bold in Table 1 demonstrated activation-stabilization ofthe enzyme at an unexpectedly high temperature of at least 160° F.

TABLE 1 Temp (F) Temp (F) Temp (F) Film Run Additive 1 Additive 1 EnzymeEnzyme Treatment 1 None None 800 ppm Savinase 160-170 160-170 2 400 ppmSavinase 65 400 ppm Savinase 160-170 160-170 3 400 ppm Savinase 160 400ppm Savinase 65 65 4 400 ppm Savinase 180 400 ppm Savinase 65 65 5 400ppm Savinase 180 400 ppm Savinase 180 170-180 6 None None 800 ppmEsperase 160-170 160-170 7 400 ppm Esperase 65 400 ppm Esperase 160-170160-170 8 400 ppm Savinase 65 400 ppm Esperase 160-170 160-170 9 NoneNone 800 Lipex 160-170 160-170 10 400 ppm Savinase — 800 Lipex — — 11400 ppm Gelatin 65 400 ppm Savinase 160-170 160-170 12 400 ppm Proline65 400 ppm Savinase 160-170 160-170 13 400 ppm Whey 65 400 ppm Savinase160-170 160-170 14 400 ppm CAPB 65 400 ppm Savinase 160-170 160-170 15400 ppm CAPB 65 400 ppm Esperase 160-170 160-170 16 400 ppm CAPB 180 400ppm Savinase 180 170-180 17 400 ppm NaHCO₃ 65 400 ppm Savinase 160-170160-170

Results

As shown in Table 1, control Runs 1, 6 and 9 were completed using onlythe protease enzymes (i.e. Savinase 800 ppm, Esperase 800 ppm, Lipex 800ppm, respectively) in solution. Each of the proteases were unable toremove any layers of the protein from the film at the elevatedtemperatures of 160-170° F. This is consistent with the understandingand current use of enzymes being limited to temperatures well below thetested range of 160-170° F., which is illustrative of ware washtemperature conditions under which proteases are not expected tosurvive. For example, the protease Savinase is one of the most readilydenatured commercially-available proteases. Savinase is known to quicklydenature at temperatures in excess of 60° C. (i.e. 140° F.).

In comparison, when various protease enzymes were combined with anadditive, the enzymes were able to remove the layers of protein film atthe same high temperature ranges of 160-170° F. using half theconcentration (400 ppm)—see Runs 2, 3, 8 and 12-15, demonstrating anembodiment of the invention wherein an enzyme in combination with anactivator promotes the unexpected efficacy of the enzyme at suchelevated temperatures. In particular, Runs 2, 3, 8 and 12-15 yieldedenzymes that retained enzymatic ability and/or were activated to enhanceenzymatic activity in order to remove the layers of protein on thephotographic film at elevated temperatures. In particular, these enzymeswere able to do so at temperatures of at least 160° F. Runs 2 and 14-15yielded the most efficacious removal of protein layers from thephotographic film at elevated temperatures.

The experiment demonstrates that heat-deactivated enzymes function asactivators allows the subsequent enzyme to unexpectedly function andthrive at the elevated temperatures (e.g. 160° F., Run 3). Run 3demonstrates that an initial deactivation or denaturation of enzyme (asa result of heating to temperature of 160° F.) that is cooled andcombined with an active enzyme allows the functioning of the enzyme at atemperature of 65° F.

Experiment 2 shows a when the enzyme was added to a beaker of water at65° F. As with all the experiments, the solution in the beaker was thenheated (typically to 160° F.), which effectively “deactivates” theenzyme, and then second enzyme source added. Accordingly, the resultsdemonstrate that the use of a heat-deactivated enzyme may serve as anactivator for additional enzyme.

According to the results, the other additives that function to activateand/or stabilize enzymes at elevated temperatures (160-170° F.) includeproline (amino acid), whey (protein) and cocoamidyl propyl betaine(CAPB) (zwitterionic surfactant). These additives are 25 illustrative ofamino acids, proteins and zwitterionic surfactants, respectively, whichare suitable for use as enzyme activators/stabilizers according to theinvention.

The inventions being thus described, it will be obvious that the samemay be varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the inventions and all suchmodifications are intended to be included within the scope of thefollowing claims.

What is claimed:
 1. A method of cleaning a hard surface comprising:contacting the hard surface with an aqueous composition comprising fromabout 1 ppm to about 800 ppm protease enzyme, and about 400 ppm organicactivator-stabilizers, wherein said organic activator-stabilizersincrease stabilization of said enzyme at temperatures in excess of atleast 160° F. up to temperatures of 180° F.; and wherein said organicactivator-stabilizers are selected from the group consisting ofamphoteric amino acids consisting of proline, amphoteric proteinsconsisting of whey, and combinations of the same; and wherein thecomposition is free of additional enzyme stabilizers.
 2. The method ofclaim 1, wherein said protease in said aqueous composition is betweenabout 1 ppm and about 400 ppm.
 3. The method of claim 1, wherein saidcontacting step comprises either adfixing a multi-use solid concentratedcomposition capable of forming said aqueous composition to the inside ofa cleaning application and contacting a water source, or adding a singleuse concentrated liquid or solid composition capable of forming saidaqueous composition to a water source to form said aqueous composition.4. A method of cleaning a hard surface comprising: (a) contacting thehard surface with sufficient organic activator-stabilizer to form anaqueous composition of about 400 ppm organic activator-stabilizer,wherein said organic activator stabilizer is selected from the groupconsisting of amphoteric amino acids consisting of proline, amphotericproteins consisting of whey, and combinations of the same; and (b)contacting the hard surface with sufficient enzyme to generate fromabout 0.1 ppm to about 400 ppm protease enzyme in said aqueouscomposition, and wherein the composition is free of additional enzymestabilizers, and wherein said organic activator-stabilizer increasesstabilization of said enzyme at temperatures in excess of at least 160°F. up to temperatures of 180° F.
 5. The method of claim 4, wherein saidaqueous composition further comprises a detergent composition.
 6. Themethod of claim 4, wherein said hard surface is selected from the groupconsisting of textiles, ware, health care instruments and combinationsof the same.
 7. The method of claim 4, wherein said aqueous compositioncomprises between about 1 ppm and about 400 ppm protease enzyme.
 8. Anenzyme stabilizing and activating aqueous composition for performance atelevated temperatures comprising: (a) an aqueous composition; (b) fromabout 1 ppm to about 800 ppm protease enzyme; and (c) about 400 ppmorganic activator-stabilizers selected from the group consisting ofamphoteric amino acids consisting of proline, amphoteric proteinsconsisting of whey, and combinations of the same, and wherein saidorganic activator-stabilizers increase stabilization of said enzyme attemperatures in excess of at least 160° F. up to temperatures of 180°F.; and wherein the composition is free of additional enzymestabilizers.
 9. The composition of claim 8, wherein said organicactivator-stabilizer is amphoteric proteins consisting of whey.
 10. Thecomposition of claim 8, wherein the protease in said aqueous compositionis between about 1 ppm and about 400 ppm.
 11. The composition of claim 8further comprising a detergent.